三叶木通MSAP反应体系的优化及引物筛选

目的研究三叶木通表观遗传多样性,建立并优化三叶木通甲基化敏感扩增多态性(methylation sensitive amplifiedpolymorphism,MSAP)反应体系。方法以三叶木通叶片为材料,利用正交试验建立三叶木通MSAP的预扩增和选择性扩增的最佳反应体系。结果建立的MSAP最佳反应体系为酶切反应:HpaII/MspI 10 U,EcoR I 10 U;预扩增反应:总体积20μL,连接产物2μL,E00、HM00各125 ng,dNTPs 0.312 5 mmol/L,Mg2+1.875 mmol/L,Taq DNA聚合酶2 U;选择性扩增反应:总体积20μL,稀释200倍的预扩增产物5μL,引物E-ACT、HM-CAT各50 ng,dNTPs 0.250 mmol/L,Mg2+1.250mmol/L,Taq DNA酶1 U。利用优化的体系,最终从MSAP的80对引物中筛选出有效引物6对。结论建立的MSAP反应体系可用于后续三叶木通DNA甲基化的相关研究。

Optimization of MSAP reaction system for Akebia trifoliata and primers screening

Objective To establish and optimize the MSAP reaction system for analysis on epigenetic diversity in Akebia trifoliata.Methods The leaves of A.trifoliata were as materials,using orthogonal L16(45) method to establish pre-amplification and selective amplification optimum reaction system of the MSAP.Results The optimal MSAP reaction systems include: 10 U HpaII/MspI and EcoR I in the enzyme digestion;in the 20 μL pre-amplication mixture contained 2 μL of ligation products,125 ng of each pre-selective primers,E00 and HM0;0.312 5 mmol/L of dNTPs,1.875 mmol/L of Mg2+,2 U of Taq DNA polymerase;in the 20 μL selective amplification mixture contained 5 μL of 1∶200 diluted pre-amplification products,50 ng of each selective primer,0.250 mmol/L of dNTPs,1.250 mmol/L of Mg2+,and 1 U of Taq DNA polymerase.Using the optimal MSAP reaction system to screen for six pairs of effective primers from 80 pairs of primers of MSAP.Conclusion Those results provide fundamental reference for further epigenetic studies on A.trifoliata DNA methylation.